Apparatus and methods for active biological sample preparation

ABSTRACT

Systems and methods for the electronic sample preparation of biological materials utilize the differential charge-to-mass ratio and/or the differential affinity of sample constituents to separation materials for sample preparation. An integrated system is provided for performing some or all of the processes of: receipt of biological materials, cell selection, sample purification, sample concentration, buffer exchange, complexity reduction and/or diagnosis and analysis. In one embodiment, one or more sample chambers adapted to receive a buffer solution are formed adjacent to a spacer region which may include a trap or other affinity material, electrophoretic motion of the materials to be prepared being effected through operation of electrodes. In another aspect of this invention, a transporter or dipstick serves to collect and permit transport of materials, such as nucleic acids, most preferably DNA and/or RNA. In one embodiment, a membrane or trap is held in a frame which is adapted to mate with a channel formed in the spacer region. In another aspect of this invention, an electrophoretic system for biological sample preparation is operated in a manner so as to utilize the differential charge-to-mass ratio so as to control the migration of materials within the solution. In one aspect, bunching of selected materials is achieved by operation of two electrodes in a manner so as to reduce the spatial dispersion of those materials. In another aspect of this invention, a vertically disposed sample preparation unit includes an upper reservoir including and a collection chamber. A sample is preferably pre-prepared and densified, applied to the conductive polymer, electrophoresed so as to move nucleic acids into the conductive polymer and move undesired material away from the conductive polymer. Integrated systems are described in which cell separation, purification, complexity reduction and diagnosis may be performed together. In the preferred embodiment, cell separation and sample purification are performed in a first region, the steps of denaturation, complexity reduction and diagnosis being performed in a second region.

FIELD OF THE INVENTION

This invention relates to devices and methods for performing active,multi-step molecular and biological sample preparation and diagnosticanalyses. More particularly, the invention relates to samplepreparation, cell selection, biological sample purification, complexityreduction, biological diagnostics and general sample preparation andhandling.

BACKGROUND OF THE INVENTION

Molecular biology comprises a wide variety of techniques for theanalysis of nucleic acid and protein. Many of these techniques andprocedures form the basis of clinical diagnostic assays and tests. Thesetechniques include nucleic acid hybridization analysis, restrictionenzyme analysis, genetic sequence analysis, and the separation andpurification of nucleic acids and proteins (See, e.g., J. Sambrook, E.F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2Ed., Cold spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989).

Most of these techniques involve carrying out numerous operations (e.g.,pipetting, centrifugations, electrophoresis) on a large number ofsamples. They are often complex and time consuming, and generallyrequire a high degree of accuracy. Many a technique is limited in itsapplication by a lack of sensitivity, specificity or reproducibility.For example, these problems have limited many diagnostic applications ofnucleic acid hybridization analysis.

The complete process for carrying out a DNA hybridization analysis for agenetic or infectious disease is very involved. Broadly speaking, thecomplete process may be divided into a number of steps and substeps. Inthe case of genetic disease diagnosis, the first step involves obtainingthe sample (e.g., blood or tissue). Depending on the type of sample,various pre-treatments would be carried out. The second step involvesdisrupting or lysing the cells, which then releases the crude DNA andRNA (for simplicity, a reference to DNA in the following text alsorefers to RNA, where appropriate) material along with other cellularconstituents. Generally, several sub-steps are necessary to remove celldebris and to purify further the crude lysate. At this point severaloptions exist for further processing and analysis. One option involvesdenaturing the purified sample DNA and carrying out a directhybridization analysis in one of many formats (dot blot, microbead,microtiter plate, etc.). A second option, called Southern blothybridization, involves cleaving DNA with restriction enzymes,separating the DNA fragments on an electrophoretic gel, blotting to amembrane filter, and then hybridizing the blot with specific DNA probesequences. This procedure effectively reduces the complexity of thegenomic DNA sample, and thereby helps to improve the hybridizationspecificity and sensitivity. Unfortunately, this procedure is long andarduous. A third option is to carry out the polymerase chain reaction(PCR) or other amplification procedure. The PCR procedure amplifies(increases) the number of target DNA sequences. Amplification of targetDNA helps to overcome problems related to complexity and sensitivity inanalysis of genomic DNA or RNA. All these procedures are time consuming,relatively complicated, and add significantly to the cost of adiagnostic test. After these sample preparation and DNA processingsteps, the actual hybridization reaction is performed. Finally,detection and data analysis convert the hybridization event into ananalytical result.

The steps of sample preparation and processing have typically beenperformed separate and apart from the other main steps of hybridizationand detection and analysis. Indeed, the various substeps comprisingsample preparation and DNA processing have often been performed as adiscrete operation separate and apart from the other substeps.Considering these substeps in more detail, samples have been obtainedthrough any number of means, such as obtaining of whole blood, tissue,or other biological fluid samples. In the case of blood, the sample isoften processed to remove red blood cells and retain the desirednucleated (white) cells. This process is usually carried out by densitygradient centrifugation. Cell disruption or lysis is then carried out,preferably by the technique of sonication, freeze/thawing, or byaddition of lysing reagents.

In certain cases, the blood is extensively processed to removecontaminants. One such system known to the prior art is the Qiagensystem. This system involves prior lysis followed by digestion withproteinase K, after which the sample is loaded onto a column and theneluted with a high salt buffer (e.g., 1.25 M NaCl). The sample isconcentrated by precipitation with isopropanol and then centrifuged toform a pellet. The pellet is then washed with ethanol and centrifuged,after which it is placed in a desired buffer. The total purificationtime is greater than approximately two hours and the manufacturer claimsan optical density ratio (260 nm/280 nm) of 1.7 to 1.9 (OD 260-280). Thehigh salt concentration can preclude performance of certain enzymaticreactions on the prepared materials. Further, DNA prepared by the Qiagenmethod has relatively poor transport on an electrophoretic diagnosticsystem using free field electrophoresis.

Returning now to the general discussion of sample preparation, crude DNAis often separated from the cellular debris by a centrifugation step.Prior to hybridization, double-stranded DNA is denatured intosingle-stranded form. Denaturation of the double-stranded DNA hasgenerally been performed by the techniques involving heating (>Tm),changing salt concentration, addition of base (e.g., NaOH), ordenaturing reagents (e.g., urea, formamide). Workers have suggesteddenaturing DNA into its single-stranded form in an electrochemical cell.The theory is stated to be that there is electron transfer to the DNA atthe interface of an electrode, which effectively weakens thedouble-stranded structure and results in separation of the strands. See,e.g., Stanley, "DNA Denaturation by an Electric Potential", U.K. patentapplication 2,247,889 published Mar. 18, 1992.

Nucleic acid hybridization analysis generally involves the detection ofa very small number of specific target nucleic acids (DNA or RNA) withan excess of probe DNA, among a relatively large amount of complexnon-target nucleic acids. DNA complexity is sometimes overcome to somedegree by amplification of target 30 nucleic acid sequences usingpolymerase chain reaction (PCR). (See, M. A. Innis et al, PCR Protocols:A Guide to Methods and Applications, Academic Press, 1990). Whileamplification results in an enormous number of target nucleic acidsequences that improves the subsequent direct probe hybridization step,amplification involves lengthy and cumbersome procedures that typicallymust be performed on a stand alone basis relative to the other substeps.Complicated and relatively large equipment is required to perform theamplification step.

The actual hybridization reaction represents an important step andoccurs near the end of the process. The hybridization step involvesexposing the prepared DNA sample to a specific reporter probe, at a setof optimal conditions for hybridization to occur to the target DNAsequence. Hybridization may be performed in any one of a number offormats. For example, multiple sample nucleic acid hybridizationanalysis can be conducted on a variety of filter and solid supportformats (See, G. A. Beltz et al., in Methods in Enzymology, Vol. 100,Part B, R. Wu, L. Grossman, K. Moldave, Eds., Academic Press, New York,Chapter 19, pp. 266-308, 1985). One format, the so-called "dot blot"hybridization, involves the non-covalent attachment of target DNAs to afilter, which are subsequently hybridized with a radioisotope labelledprobe(s). "Dot blot" hybridization has gained wide-spread use, and manyversions have been developed (See, M. L. M. Anderson and B. D. Young, inNucleic Acid Hybridization--A Practical Approach, B. D. Hames and S. J.Higgins, Eds., IRL Press. Washington, D.C. Chapter 4, pp. 73-111, 1985)."Dot blot" assays have been developed for the multiple analysis ofgenomic mutations (D. Nanibhushan and D. Rabin, in EPA 0228075, Jul. 8,1987) and for the detection of overlapping clones and the constructionof genomic maps (G. A. Evans, in U.S. Pat. No. 5,219,726, Jun. 15,1993).

New techniques are being developed for carrying out multiple samplenucleic acid hybridization analysis on micro-formatted multiplex ormatrix devices (e.g., DNA chips) (See, M. Barinaga, 253 Science, pp.1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). Thesemethods usually attach specific DNA sequences to very small specificareas of a solid support, such as micro-wells of a DNA chip. Thesehybridization formats are micro-scale versions of the conventional "dotblot" and "sandwich" hybridization systems.

The micro-formatted hybridization can be used to carry out "sequencingby hybridization" (SBH) (See, M. Barinaga, 253 Science, pp. 1489, 1991;W. Bains, 10 Bio/Technology, pp. 757-758, 1992). SBH makes use of allpossible n-nucleotide oligomers (n-mers) to identify n-mers in anunknown DNA sample, which are subsequently aligned by algorithm analysisto produce the DNA sequence (R. Drmanac and R. Crkvenjakov, YugoslavPatent Application #570/87, 1987; R. Drmanac et al., 4 Genomics, 114,1989; Strezoska et al., 88 Proc. Natl. Acad. Sci. USA 10089, 1992; andR. Dramanac and R. B. Crkvenjakov, U.S. Pat. No. #5,202,231, Apr. 13,1993).

There are two formats for carrying out SBH. The first format involvescreating an array of all possible n-mers on a support, which is thenhybridized with the target sequence. The second format involvesattaching the target sequence to a support, which is sequentially probedwith all possible n-mers. Both formats have the fundamental problems ofdirect probe hybridizations and additional difficulties related tomultiplex hybridizations.

Southern, United Kingdom Patent Application GB 8810400, 1988; E. M.Southern et al., 13 Genomics 1008, 1992, proposed using the first formatto analyze or sequence DNA. Southern identified a known single pointmutation using PCR amplified genomic DNA. Southern also described amethod for synthesizing an array of oligonucleotides on a solid supportfor SBH. However, Southern did not address how to achieve an optimalstringency condition for each oligonucleotide on an array.

Concurrently, Drmanac et al., 260 Science 1649-1652, 1993, used thesecond format to sequence several short (116 bp) DNA sequences. TargetDNAs were attached to membrane supports ("dot blot" format). Each filterwas sequentially hybridized with 272 labelled 10-mer and 11-meroligonucleotides. A wide range of stringency conditions was used toachieve specific hybridization for each n-mer probe; washing timesvaried from 5 minutes to overnight, and temperatures from 0° C. to 16°C. Most probes required 3 hours of washing at 16° C. The filters had tobe exposed for 2 to 18 hours in order to detect hybridization signals.The overall false positive hybridization rate was 5% in spite of thesimple target sequences, the reduced set of oligomer probes, and the useof the most stringent conditions available.

A variety of methods exist for detection and analysis of hybridizationevents. Depending on the reporter group (fluorophore, enzyme,radioisotope, etc.) used to label the DNA probe, detection and analysisare carried out fluorometrically, colorimetrically, or byautoradiography. By observing and measuring emitted radiation, such asfluorescent radiation or particle emission, information may be obtainedabout the hybridization events. Even when detection methods have veryhigh intrinsic sensitivity, detection of hybridization events isdifficult because of the background presence of non-specifically boundmaterials. A number of other factors also reduce the sensitivity andselectivity of DNA hybridization assays.

Attempts have been made to combine certain processing steps or substepstogether. For example, various microrobotic systems have been proposedfor preparing arrays of DNA probes on a support material. For example,Beattie et al., in The 1992 San Diego Conference: Genetic Recognition,November, 1992, used a microrobotic system to deposit micro-dropletscontaining specific DNA sequences into individual microfabricated samplewells on a glass substrate. Various attempts have been made to describeintegrated systems formed on a single chip or substrate, whereinmultiple steps of an overall sample preparation and diagnostic systemwould be included. For example, A. Manz et al., in "Miniaturized TotalChemical Analysis System: A Novel Concept For Chemical Sensing", SensorsAnd Actuators, B1(1990), pp. 244-248, describe a `total chemicalanalysis system` (TAS) which comprises a modular construction of aminiaturized total chemical analysis system. Sampling, sample transport,any necessary chemical reactions, chromatographic separations as well asdetection were to be automatically carried out. Yet another proposedintegrated system is Stapleton, U.S. Pat. No. 5,451,500, which describesa system for the automated detection of target nucleic acid sequences inwhich multiple biological samples are individually incorporated intomatrices containing carriers in a 2-dimensional format. Different typesof carriers are described for different kinds of diagnostic tests ortest panels.

Various multiple electrode systems are disclosed which purport toperform multiple aspects of biological sample preparation or analysis.Pace, U.S. Pat. No. 4,908,112, entitled "Silicon Semiconductor Wafer forAnalyzing Micronic Biological Samples" describes an analyticalseparation device in which a capillary-sized conduit is formed by achannel in a semiconductor device, wherein electrodes are positioned inthe channel to activate motion of liquids through the conduit. Pacestates that the dimension transverse to the conduit is less than 100 μm.Pace states that all functions of an analytical instrument may beintegrated within a single silicon wafer: sample injection, reagentintroduction, purification, detection, signal conditioning circuitry,logic and on-board intelligence. Soane et al., in U.S. Pat. No.5,126,022, entitled "Method and Device for Moving Molecules by theApplication of a Plurality of Electrical Fields", describes a system bywhich materials are moved through trenches by application of electricpotentials to electrodes in which selected components may be guided tovarious trenches filled with antigen-antibodies reactive with givencharged particles being moved in the medium or moved into contact withcomplementary components, dyes, fluorescent tags, radiolabels,enzyme-specific tags or other types of chemicals for any number ofpurposes such as various transformations which are either physical orchemical in nature. It is said that bacterial or mammalian cells, orviruses may be sorted by complicated trench networks by application ofpotentials to electrodes where movement through the trench network ofthe cells or viruses by application of the fields is based upon thesize, charge or shape of the particular material being moved. Clark,U.S. Pat. No. 5,194,133, entitled "Sensor Devices", discloses a sensordevice for the analysis of a sample fluid which includes a substrate ina surface of which is formed an elongate micro-machined channelcontaining a material, such as starch, agarose, alginate, carrageenan orpolyacrylamide polymer gel, for causing separation of the sample fluidas the fluid passes along the channel. The biological material maycomprise, for example, a binding protein, an antibody, a lectin, anenzyme, a sequence of enzymes or a lipid.

Various devices for eluting DNA from various surfaces are known. ShuklaU.S. Pat. No. 5,340,449, entitled "Apparatus for Electroelution"describes a system and method for the elution of macromolecules such asproteins, DNA and RNA from solid phase matrix materials such aspolyacrylamide, agarose and membranes such as PVDF in an electric field.Materials are eluted from the solid phase into a volume defined in partby molecular weight cut-off membranes. Okano, U.S. Pat. No. 5,434,049,entitled "Separation of Polynucleotides Using Supports Having aPlurality of Electrode-Containing Cells" discloses a method fordetecting a plurality of target polynucleotides in a sample, the methodincluding the step of applying a potential to individual chambers so asto serve as electrodes to elute captured target polynucleotides, theeluted material then available for collection.

Generally, the prior art processes have been extremely labor and timeintensive. For example, the PCR amplification process is time consumingand adds cost to the diagnostic assay. Multiple steps requiring humanintervention either during the process or between processes issuboptimal in that there is a possibility of contamination and operatorerror. Further, the use of multiple machines or complicated roboticsystems for performing the individual processes is often prohibitiveexcept for the largest laboratories, both in terms of the expense andphysical space requirements.

As is apparent from the preceding discussion, numerous attempts havebeen made to provide effective techniques to conduct sample preparationreactions. However, for the reasons stated above, these techniques arelimited and lacking. These various approaches are not easily combined toform a system which can carry out a complete DNA diagnostic assay.Despite the long-recognized need for such a system, no satisfactorysolution has been proposed previously.

SUMMARY OF THE INVENTION

This invention relates broadly to the methods and apparatus forelectronic sample preparation of biological materials for their ultimateuse in diagnosis or analysis. An integrated system is provided forperforming some or all of the processes of: receipt of biologicalmaterials, cell selection, sample purification, complexity reductionand/or diagnosis and analysis. Separation of desired components, such asDNA, RNA or proteins from crude mixtures such as biological materials orcell lysates. Electronic sample preparation utilizes the differentialmobility and/or differential affinity for various materials in thesample for purposes of preparation and separation. These methods andapparatus are especially useful for the free field electrophoreticpurification of DNA from a crude mixture or lysate. In one aspect ofthis invention, a device comprises at least a first central or samplechamber adapted to receive a buffer solution and a second central orsample chamber adapted to receive a same or different buffer solution,where the first sample chamber and the second sample chamber areseparated by a spacer compartment which preferably includes a trap,membrane or other affinity material. A first electrode in electricalcontact with the conductive solution of the first sample chamber and asecond electrode in electrical contact with the conductive solution ofthe second sample chamber are provided. Preferably, each electrode iscontained within its own electrode chamber, the electrode chamber beingseparated from the corresponding sample chamber via a protective layeror separation medium, e.g., a membrane, such as an ultrafiltrationmembrane, a polymer or a gel. In operation, the sample mixture is placedwithin the first central chamber. The central chambers contain asolution, preferably a low conductivity buffer solution, such as 50 mMhistidine, 250 mM HEPES, or 0.5×TBE. A mixture of biological substances,for example a crude lysate, is added to the first chamber and then theelectrodes are activated. Substances with mobility in an electric fieldwill move towards one electrode or the other depending on the charge ofthe substance. In one embodiment, the desired and undesired substanceswith similar charges will be attracted to an electrode biased with theopposite charge to the desired substance, located in the second chamber.Therefore, the desired substance and the undesired substances of similarcharge will move towards the affinity material which is between thefirst and second chambers.

In one embodiment, the sample mixture is composed of a desired substancewhich has charge mixed with undesired substances some of which havecharges. After the electrodes are activated, the desired substancetravels toward the second chamber where the electrode, biased withopposite charge, is located and binds to the affinity material. Incontrast, the similarly charged, undesired substances move toward theelectrode biased with the opposite charge and pass through the affinitymaterial into the second chamber. Other undesired substances with theopposite charge will be attracted toward the electrode in the firstchamber. After the undesired substances have passed through the affinitymaterial, the electrolyte solution may be changed in both chambers toremove the undesired substances. Then the desired substance is elutedinto the fresh electrolyte solution. Elution can be accomplished bycontinued electrophoresis at the same or increased current or by theaddition of a chemical, such as a detergent, salt, a base or an acid,that will cause elution from the affinity material. In addition, achange in temperature could be used to elute the desired substance.

In one particular embodiment, the affinity material is composed of a gelwith sufficient volume to hold a substantial fraction, e.g., preferably50%, and more preferably, 80%, of the desired substance in the sample.The gel composition and concentration is chosen such that the mobilityof the desired substance which has high molecular weight (30,000 to3,000,000 daltons) is retarded by the gel but the mobility of theundesired substances which have low molecular weight (100 to 10,000daltons) is relatively unaffected. Consequently, the gel will releasethe desired substance only after a longer period of electrophoresis or ahigher current than is necessary for the passage of the undesiredsubstances. In effect the gel is a trap for the desired substance butdoes not provide a relatively significant barrier to undesiredsubstances. The desired and undesired substances preferably have a verylarge difference in electrophoretic mobility while traveling through thegel for the gel to serve as a trap. Preferably, the trap does noteffectively resolve mobility differences among fragments which havesimilar compositions and molecular weights which are within a factor ofsubstantially 10 of each other.

In accordance with this invention, the resolution of different molecularweights is preferentially sacrificed in favor of rapid mobility.Preferably, the gel in the device is relatively compact (e.g., 0.5 to 10mm) in the direction of migration to permit rapid electrophoretictransport. Thus, the speed of this technique is not compatible withconventional resolution of substances by size except between substanceswith very gross differences in size. Consequently, it is inherent inthis technique that desired substances of very different molecularweights will be copurified. The preparation of substances of similarcomposition but different sizes may be advantageous to the user as forexample in the purification of DNA of different sizes for the purpose ofcloning of a whole or representative portion of a genome of an organism.

In accordance with one aspect of this invention, the desired substanceis propelled into and out of the gel in the same device. That is, in thepreferred embodiment, different regions within the whole system serve astraps and therefore, help to separate analytes or materials to beeluted. The integration of electrophoresis and elution steps alsoprovides significant advantages for the user in saving time, reducingthe number of steps required and decreasing the amount of space requiredfor the apparatus.

In one aspect of the invention, a device containing multiple electrodechambers in electrical communication with a first and second end samplechamber and one or more intermediate sample chambers is advantageouslyutilized. In the preferred embodiment, each of the end sample chambersand the intermediate sample chamber or chambers is in electricalcommunication with an electrode chamber, preferably having the samplechamber separated from the electrode chamber by a membrane. Preferably,the electrode chamber has a buffer volume which is larger than andpreferably much larger than (e.g., at least 10 to 1) the volume of thesample which is loaded into the sample chamber.

In another aspect of this invention, the traps, membranes or otheraffinity materials serve as a transporter or `dipstick` to collect andpermit transport of materials. In one embodiment of this device, themembrane or other affinity material disposed between adjacent samplechambers is provided with structural integrity to permit the removal ofthe trap or affinity material from the chamber structures. In onepreferred embodiment, a membrane or trap is held in a frame which isadapted to mate with a channel formed at the spacer region. Thetransporter or dipstick may be utilized to transport the collectedmaterial for further processing, such as further purification,complexity reduction or assaying or diagnosis. Optionally, thetransporter may be disposed adjacent to or formed in electricalcommunication with a power source.

In yet another aspect of this invention, first and second electrodes aredisposed within an intervening sample solution, the system furtherincluding a third electrode between the first and second electrodes. Thethird electrode may serve as a control electrode to modulate the flow ofcharged materials within the sample solution. The third electrode ispreferably formed as a grid, and may be advantageously formed bysputtering a metal coating on a membrane. The third electrode, or grid,is preferably disposed within the sample solution closer to the firstelectrode than the second electrode. In one mode of operation, a sampleis placed within the sample solution between the first and thirdelectrodes, and the first and second electrodes are biased for netmigration of charged materials of a first charge toward the secondelectrode. The third electrode or grid is preferably biased slightlynegative or neutral. Once the desired DNA or other charged materialspasses through or by the third electrode or grid, the third electrode orgrid is preferably made relatively negative. This increased negativityserves to move the negatively charged DNA towards the second electrode,and to repel other, more slowly moving negatively charged materialswhich still remain between the first electrode and the third or gridelectrode.

In yet another aspect of this invention, a pair of electrodes arelocated within a sample solution and are operated so as to bunch orconcentrate a subset of the charged macromolecules within the samplesolution. Biasing one electrode so as to accelerate motion of chargedmacromolecules towards the other electrode, and biasing the otherelectrode so as to retard motion of the charged macromolecules which arecloser to that electrode in the region between the electrodes. Suchbunching serves to physically concentrate the charged macromoleculeswithin the region.

In a preferred embodiment, a "C" shaped electrode is utilized. Thisstructure serves to bunch materials contained within the region boundedby the C-shaped electrode, as well as to repel like charged materialswhich are external to the region bounded by the C-shaped region.Further, the materials contained within the C-shaped region are subjectto a force in a sideways (i.e., a transverse or oblique direction to thenet flow direction apart from the C-shaped electrode). The C-shapedelectrode may be an integrated, continuous electrode or may besegmented. Other shapes are advantageously utilized, such as parabolicstructures. The C-shaped region is sized to include within the regionthe desired or target material.

A complexity reduction device includes one or more probe areas whichcomprise a support material, preferably a polymer gel, such as anagarose, acrylamide or other conductive polymer, to which capture probesare attached. The support material is formed in contact with anelectrode to permit the electrophoretic attraction and hybridization ofthe capture probes with the target materials. Electrical elution orelectronic stringency control of the captured materials may be used. Inone embodiment, the complexity reduction device is formed from thecombination of a chamber mated to a printed circuit board. The chamberincludes vias in which the support material is located. The printedcircuit board preferably including concentric vias to provide acontinuous space for the inclusion of the support material. In thisembodiment, gases or other reaction products may be vented through thevia, in part because the gas generally does not rise into the via sinceit is filled with polymer. Therefore, these gases or other reactionproducts do not come in contact with the analytes, such as DNA. Thecomplexity reduction system optionally may further include disposalregions for the attraction and/or disposal of undesired materials. Thedisposal regions include an electrode in electrical communicationthrough the conductive polymer. In operation, the complexity reductiondevice performs free field electrophoretic transport. Alternatively, anuncovered electrode in contact with the solution may be utilized fordisposal of undesired materials.

In yet another aspect of this invention, a DNA or other nucleic acidpurification device is provided. An upper reservoir containing anelectrode, which may be identified as a cathode, is adapted to receive abuffer solution and a sample solution. Preferably, the upstreamreservoir includes a tube in fluid communication with the upstreamreservoir, the tube having an internal diameter less than the diameterof the upstream reservoir. The tube includes at least a firstdifferential mobility section, preferably a gel, which provides a plugor trap region within the tube. Optionally, the gel may be cast on topof a support membrane. A collection chamber is adjacent to thedifferential mobility region. In the preferred embodiment, thecollection region has a smaller volume than the differential mobilityregion and is smaller than the sample volume. The reduction in volumefrom sample to collection permits an increase in volume concentration ofDNA and exchanges the DNA into the desired buffer formulation of knownvolume. An anode is provided in a lower reservoir. In yet another aspectof this invention, the format and transport of DNA is in the horizontalplane instead of vertical.

In operation, a sample is subject to a cell lysing and shearingpre-preparation step. Preferably, the proteins are then reduced in size,such as through application of a proteinase, such as proteinase K.Preferably, when the unit is operated in a vertical format, sucrose orother densifier is added to the sample. The densifier serves to collectand concentrate the sample in a region immediately above the firstdifferential mobility section. The prepared sample including densifieris then injected onto the separation unit in the tube, in a regionimmediately upstream of the differential mobility region. The cathodeand anode are then energized to provide electrophoretic transport of thecharged materials within the system, causing the reduced size proteinmaterials to pass through the differential separation medium first,while retaining the relatively slower moving DNA, as well as resultingin the proteinase K or other positively charged materials being removedto the cathode. After a time sufficient to permit desired amounts ofprotein to pass through the differential mobility region and supportmembrane, the DNA is eluted from the differential movement region intothe sample chamber. Optionally, the buffer solution in the lowerreservoir which received the proteinaceous material having passedthrough the support membrane may be removed and replaced with new buffersolution, and optionally provided with a membrane which serves to retaineluted DNA within the sample chamber.

In yet another aspect of this invention, the cathode and anodeelectrodes are in communication with the sample through conductivefluids or gel or polymers, but are resident in the power supply or othercontrolling instrumentation. Conduction is made through fluidic portsand the electrode (noble metal) preferably is not a part of theconsumable device.

Integrated systems may be advantageously formed which include some orall of the functions of cell separation, purification, complexityreduction and diagnostics. In one embodiment, a purification chamberincludes an input port and multiple electrodes for providing anelectrophoretic driving force to the charged materials. A protein trapdisposed between the sample input port and an outlet tap serves to trapundesired proteins or other charged macromolecules. In an alternativeembodiment, the undesired materials such as proteins are reduced in sizeor modified in charge so as to increase their degree of mobilityrelative to the desired materials, such as DNA. The materials forfurther processing are then removed from the trap or othertransportation device for further processing, such as complexityreduction and/or diagnostic procedures. This separation of targetmaterials from the undesired material is accomplished by modifying theelectric field to steer the target materials into a region of higherpurity (e.g., pure buffer). Electric field modification can beimplemented through C-shape electrodes or activation of a newconfiguration of electrodes which favorably influence the direction ofmobility of the target.

In yet another aspect of this invention, an integrated samplepreparation, complexity reduction and diagnostic system is provided. Aninput chamber receives crude lysate including proteins which have beenreduced in size, such as through use of a proteinase. The materials passthrough a DNA trap, in which the DNA moves relatively slower than theproteins. The proteins arrive at a protein trap electrophoreticallyprior to the arrival of the DNA. The DNA then exit the DNA trap towardsa collection chamber. Preferably, the collection chamber has a volumewhich is less than, preferably substantially less than the volume of theDNA trap, such as 50 microliters. This volume is in communication withthe complexity reduction and diagnostic chambers. In one aspect of thisinvention, fluidic transport within the device is accomplished by inputports located at each end of the collection chamber. These dual inputports are adapted to receive any fluid, such as a buffer, an air slug ora reagent. By selective operation of supplies or pumps, the materialscontained within the collection chamber, e.g., substantially purifiedDNA, may be forced from the chamber to the complexity reduction device.By utilization of air slugs, various liquids may be separated from oneanother. By operation of the input liquids or gas, a hydraulic orpneumatic ram serves to move materials within the fluid section of thedevice. While materials may be pushed forward throughout the system,such as from the collection volume to the complexity reduction to thediagnostic chamber, the materials may be moved in the oppositedirection, e.g., from the complexity reduction chamber to theconcentration chamber.

Accordingly, it is an object of this invention to provide for a samplepreparation system useful for biological sample preparation.

It is yet a further object of this invention to provide a system forpurification of DNA from biological materials or other crude samples.

It is yet a further object of this invention to provide methods for theseparation and purification of desired biological charged macromoleculesthrough differential solution phase mobility and/or differentialaffinity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top, plan view of a multiple sample chamber, multipleelectrode chamber device.

FIG. 2 is a top, plan view of a device including multiple electrodechambers, end sample chambers and multiple intermediate sample chambers.

FIG. 3 is a perspective, exploded view of one embodiment of theinvention.

FIG. 4 is a cross-sectional view of a multiple electrode embodiment.

FIG. 5 is a cross-sectional view of a multiple electrode embodimentincluding a trap electrode.

FIG. 6 is a perspective view of a multiple electrode structure includinga grid or control electrode.

FIG. 7 is a perspective view of a multiple electrode system furtherincluding bunching electrodes.

FIG. 8 is a plan view of one implementation of an integrated samplepreparation and diagnostic device.

FIG. 9 is a plan view of another integrated sample preparation anddiagnostic device.

FIG. 10 is a perspective, cross-sectional view of the complexityreduction device.

FIG. 11 is a perspective, cross-sectional close-up view of thecomplexity reduction device.

FIG. 12 is a perspective view of the complexity reduction device withthe printed circuit board and complexity reduction chamber exploded fromeach other.

FIG. 13 is a cutaway perspective drawing of a vertically disposed samplepreparation device.

FIG. 14 shows a plan view of a horizontal integrated sample preparation,complexity reduction, diagnostic and disposal device.

FIG. 14A shows a cross-sectional view along the line A-A' of FIG. 14.

FIG. 15 is a graph of target DNA transport in the presence of S. aureusGenomic DNA purified by the device of FIG. 13 compared to the prior artQiagen method, as a function of time.

FIG. 16 is a graph comparing the operation of the complexity reductiondevice in different buffer solutions.

FIG. 17 is a graph of the signal accumulation for a microelectronicdevice in comparison to a printed circuit board based device for variousbuffers.

FIG. 18 is a graph of relative target signal levels determined forspecific and nonspecific probes following transport and electronic wash,and after dehybridization using a Texas Red Bodipy™ labelledStreptococcal target sequence in the presence of irrelevant human DNAper 40 μL.

FIG. 19 is a graph of hybridization of labeled Streptococcal target DNAin the presence of an equimolar concentration of nonlabeledcomplementary DNA and irrelevant human DNA.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows a top down, plan view of one embodiment of this invention.A frame 10 supports a first central or sample chamber 12 and a secondcentral or sample chamber 14. The designations first and second arearbitrary, and may be reversed, or the chambers may be referred to asthe left central chamber 12 and right central chamber 14. Disposedbetween the left central chamber 12 and right central chamber 14 is aspacer compartment 16. The spacer compartment is adapted to receive atrap, membrane or other affinity material or material for relativedifferentiation of biological materials within the spacer compartment16. In one aspect, the spacer compartment 16 may be filled with a gelcast in the chamber of the spacer compartment 16 or a membrane may bedisposed covering the spacer compartment 16. Within the left centralchamber 12 is a first electrode chamber 18. The electrode chamber 18 isseparated from the central chamber 12 by a membrane 22, preferably anultrafiltration membrane, most preferably a cellulose acetate membrane.Similarly, a right or second electrode chamber 20 is disposed adjacentto the right central chamber 14, separated by a membrane 24. A functionof the membranes 22, 24 is to reduce or prevent contact of the samplematerials or selected components thereof from directly contacting theelectrodes 26, 28. A first electrode 26 and a second electrode 28 aredisposed in contact with the first electrode chamber 18 and secondelectrode chamber 20, respectively. The electrodes are preferably formedof a noble metal, most especially platinum.

In the preferred embodiment, the frame 10 is formed of a relativelyrigid support material which is non-reactive with the materials placedin contact with it. Desired materials include: polymethacrylate,plastics, polyproplene, polycarbonate, PTFE, TEFLON™ or othernon-reactive materials. Generally, the frame materials are non-reactive,non-interactive or non-binding with the sample materials. The frame 10may have formed within it chambers comprising the electrode chambers 18,20, central chambers 12, 14 and the spacer compartment 16. In oneembodiment, the electrode chambers 18, 20, are 0.4 cm wide, 0.6 cm deepand 5 cm long (the direction parallel to a line between the firstelectrode 26 and second electrode 28). The central chambers 12, 14 havethe same width and depth as the electrode chambers 18, 20 and are 1.5 cmlong. The spacer compartment 16 has the same width and depth as thechamber components 12, 14, 18 and 20, and is 0.4 cm long. Generally, thevolume of the spacer compartment 16 is small relative to the volume ofthe central chamber 12, 14. Preferably, the volume of the spacercompartment 16 is less than 50%, and more preferably less than 30% ofthe volume of the central chamber 12, 14. The embodiment described aboveas a spacer compartment 16 with a volume of approximately 25% of that ofthe central chamber 12, 14. As an alternative measure, the spacercompartment 16 may be characterized by its distance along the directionthe direction from the first electrode 26 to the second electrode 28.Preferably, the linear distance is less than 5 mm, more preferably 4 mmor less, and often in the range of 1-2 mm. In yet anothercharacterization, the length of the spacer compartment 16 in a directionbetween the first electrode 26 and the second electrode 28 is shortrelative to the distance between the first electrode 26 and secondelectrode 28. Preferably, this length is less than 20%, more preferablyless than 10% and most preferably less than approximately 5%.Preferably, the size of the spacer compartment 16 is such, relative tothe other structures of the device, that the desired materials may beseparated from the undesired materials, so as to permit isolation of thedesired materials.

The spacer compartment 16 includes a material which serves todiscriminate amongst macromolecules within the device. One class ofmaterials would comprise protein traps, materials such as polyvinylidenediflouride (PVDF), nitrocellulose and hydrophobic materials. Yet anotherclass of materials are negatively charged materials. Yet a third classof materials are positively charged materials. A modified class ofmaterials utilizes a detergent in combination with the trap whichpermits the DNA to pass through the trap. Various other surfactants,detergents and materials which achieve the functions of the trap todifferentiate amongst materials may be utilized as known to thoseskilled in the art. DNA/RNA traps would especially include low densitypolymers (e.g., 0.5-3% agarose, or 5%-15% acrylamide) and PVDF. Thelatter material is a material which traps DNA to some extent, but fromwhich DNA or RNA may be selectively eluted by adding a detergent.

The electrode chambers 18, 20 and central chambers 12, 14 are filledwith a buffer solution. Preferably, the buffer solution is a relativelylow conductivity buffer solution. Other functional characteristics ofthe buffer may include: chemically low reactivity, Zwitterionic orampholytes with no net charge. The application entitled "Methods andMaterials for Optimization of Electronic Hybridization Reactions", filedon the same date as the instant application, and incorporated herein byreference more fully describes such techniques. Examples of bufferswhich are suitably acceptable for separation of DNA include: histidine,especially about 50 millimolar histidine, HEPES and 0.5×TBE. HEPES is4(-2-Hydroxyethyl)-1-Piperazine Ethanesulfonic acid. TAE is prepared bydiluting 10×TAE (0.4 M Tris Acetate, 0.1 M EDTA, 0.2 M glacial aceticacid pH 8.4) 1 to 10 in deionized water. 0.5×TBE is prepared by dilutingTBE (0.89M Tris-Borate, 0.89 M Boric Acid, 0.02 M EDTA, pH 8.0) 1 to 20in deionized water.

In operation, a sample is placed in a central chamber 12, 14, forpurposes of discussion assumed to be the left central chamber 12. Apotential is applied across the first electrode 26 and second electrode28, permitting a current to flow through the electrode chambers 18, 20and central chambers 12, 14. The charged macromolecules in the sampleare electrophoretically moved through the left central chamber 12,towards the spacer compartment 16. If the spacer compartment 16 includesa protein trap, the DNA is moved through the spacer compartment 16 intothe right central chamber 14. Alternatively, if the spacer compartment16 includes a material designed to hold the DNA, but pass the proteinsand other undesired materials, the DNA remains on the left portion ofthe spacer compartment 16, from which it may be eluted. In the latermode of operation, the buffer in the left central chamber 12 may bereplaced prior to eluting the DNA back into the left central chamber 12.

Broadly, the method of this invention provides for active biologicalsample preparation of a sample comprising a collection of materialsincluding desired materials and undesired materials having differentialcharge-to-mass ratios, the separation being achieved in anelectrophoretic system including solution phase regions and at least onetrap having differential effect on desired materials as compared toundesired materials. The differential effect of the trap is a functionof the physical size, composition and structure of the trap, which areselected so as to selectively retain or pass either the desired orundesired material. In the preferred embodiment, the method includes thestep of providing the sample materials to a first sample chamber of thedevice. Subsequently, the sample is electrophoresed within the system toaffect net differential migration between the desired material and theundesired material whereby one of the desired or undesired materials islocated within the trap and the other material is in a sample chamber,that is, either the desired materials are substantially retained (e.g.,preferably ≧50%, and more preferably, ≧80%) in the trap, and theundesired materials are substantially not retained in the trap, or viceversa. Subsequently, the desired material are removed from the system,whereby relatively purified desired materials are prepared. If thedesired materials are the bound or trapped materials, they may beremoved in any number of ways, including but not limited to physicalremoval from the system, such as by a dipstick, subsequent removal intoa fluid or other medium, such as by moving through the trap or movingback into the chamber from which they came, especially if the buffersolution has been changed within that chamber.

FIG. 2 shows a plan view of a multichamber arrangement. A frame 30includes multiple electrode chambers 32. Each electrode chamber 32includes an electrode 34. Preferably, the electrode is formed of a noblemetal, such as platinum, and may be placed in electrical contact with abuffer solution placed within the electrode chamber 32. End samplechambers 36 sandwich one or more intermediate sample chambers 38. Theend sample chamber 36 is separated from the adjacent intermediate samplechamber 38 by a separator 40. The separator 40 may comprise an affinitymedia, a membrane, or other material which selectively differentiatesbetween passage or affinity for various biological materials. The samplechambers 36, 38 are separated from the electrode chamber 32 by amembrane 42. Preferably, the volume of the electrode chamber 32 islarger, preferably a factor of 10 times, and most preferably a factor of20 times, relative to the size of the sample chamber 36, 38. This is sosince electrosmosis through the membrane 42 may result in liquid-leveldifferences. Further, this relatively large volume ratio minimizes theion concentration gradient between the adjacent electrode chamber 32 andsample chamber 36, 38 and provides a larger buffering capacity aroundthe electrode 34 which increases pH stability which, in turn, optimizesthe working time and power (V×I) input to the sample before thedetrimental effects of the electrophoretically driven osmosis begin todominate and negatively impact the process. Yet another object of therelatively large electrode chamber 32 is that adequate spatialseparation between the chamber membrane 42 and the electrode 34 isprovided so as to prevent bubble attachment to the membrane 42, afterthe bubbles are formed at the electrode 34. It is desirable to avoidbubble contact with the membrane 42 as they obstruct the membrane andprevent conduction through them, and the bubbles may have large pHs.Further, it is desirable that the electrodes 34 have relatively largesurface area, e.g., 5-20 mm² which minimize bubble formation by reducinglocal current density near the electrode surfaces and the inherentsurface nucleation sites.

The frame 30 may be formed of any material which is non-reactive withthe materials to be placed in contact with it. Preferably, the materialsare selected to have low autofluorescence at 380 nmUV, and between 480nm and 630 nm, in order to minimize background signal duringquantitation. The sample chambers 36, 38 may be of different sizes.Preferably, the sample chamber 36, 38 is in the range from 300 μl to 3ml, most preferably 1 ml. After purification steps, the relativelypurified material may be reduced in sample volume, and the volume may beon the order of tens of microliters or less.

FIG. 3 shows a perspective, exploded view of a multichamber device. Aframe 50 has formed, such as by milling or molding, one or more endsample chambers 56, sample chambers 58 and electrode chambers 52 havingthe functions and sizes described in connection with FIG. 2. Theelectrode 54 preferably exits the electrode chamber 52 and is connectedvia a connector 86, such as a threaded connector as is known to thoseskilled in the art. Adjacent sample chambers 56, 58 are separated by amembrane holder 60. The membrane holder 60 optionally is formed ofmembrane holder halves 62 connected via connector 66. An opening 64 inthe membrane holder 60 is adapted to receive a material whichdifferentiates or discriminates the passage of biological materials,such as a membrane or affinity material. The membrane holder 60 isadapted to matingly engage with connector 66. The sample chamber 56, 58is in communication with the electrode chamber 52 via passage 68. Inthis embodiment, insert 70 threadingly engages with the frame 50 bythreading 72 in receptive threading 74. A barrel 76 includes acounterbore 78 and includes holes 80 to permit passage from theelectrode chamber 52 through the holes 80, through the counterbore 78,to the sample chamber 56, 58. The insert 70 preferably terminates at aring 82, opposite the threaded end of the insert 70, where the ring 82is adapted to sandwich a filter or membrane 84 between the ring 82 andthe bore 68 of the electrode chamber 52. The size of the sample chambers56, 58 may vary from one to another. Further, the various membraneholders 60 may be utilized, or not, providing yet an additional degreeof flexibility in determining the size of the sample chamber 56, 58.

The membrane holder 60 is removable from the frame 50. The membraneholder 60 may include membrane, mesh or beads with functional groupscovalently linked to oligonucleotides. After material is captured withinthe opening 64 of the membrane holder 60, the membrane holder 60 may beremoved from the frame 50 and the materials transported to another site.

FIG. 4 shows a cross-sectional view of an embodiment of this invention.A first or left electrode 90 and a second or right electrode 92 areadapted to provide an electrophoretic force on charged macromoleculesdisposed within the solution phase region 94. The solution phase chamber94 is shown with a dashed boundary, the physical boundary of which maybe formed through any desired support medium not inconsistent with thematerials or methods to be achieved with this invention. A trap 96 isdisposed at, near or substantially surrounding the second electrode 92.The trap 96 may be formed of the materials, and have the attributes asdescribed for the trap or spacer materials, above. Optionally, aprotective or permeable layer 98 is disposed between at least a portionof the solution phase region 94 and the first or right electrode 90. Asshown in FIG. 4, the permeable layer 98 serves to block the solutionphase region 94 from direct contact with the left electrode 90. Inoperation, a sample is placed in an input region 100, such as through aport or other opening in the device. The solution phase region 94contains a buffer or other suitable transport medium, comprised of orhaving the functions described for the solution phase, above. A sampleinitially placed in the input region 100 is electrophoretically movedtowards the trap 96 by application of potential to the electrodes 90,92. When the desired material, e.g., DNA, contacts the trap 96, thesystem is then operated so as to remove the now trapped materials. Thiscould be by removal of the trap 96 from the system, or by eluting thetrapped material from the trap 96 back into the solution phase region94. Preferably, the elution of the trapped material into the solutionphase region 94 is preceded by replacing the solution in the solutionphase region 94 with a new solution. The solution may be the same ordifferent from that previously existing.

FIG. 5 shows a cross-sectional view of an alternate embodiment of theinvention. A first electrode 110 and a second electrode 112 provideoverall electrophoretic movement of charged materials within the firstsolution phase region 114 and second solution phase region 115. Thefirst electrode 110 optionally has a first permeable layer 118 disposedat, near or substantially around the first electrode 110, so as tominimize contact of the charged macromolecules with the first electrode110. A second layer 109 is formed at, near or substantially surroundingthe second electrode 112. In one version, the second layer 109 comprisesa trap, the materials and functionality being those described above.Alternatively, the second layer 109 may comprise a second permeablelayer, adapted principally for the protection of charge macromoleculesfrom directly contacting the second electrode 112. Optionally, a trapelectrode 122 is located between the first electrode 110 and the secondelectrode 112, dividing the solution phase chamber into a first solutionphase chamber 114 and a second solution phase region 115. Further,optionally, the trap electrode 122 may include a trap 116 formed at orintegral with the trap electrode 122. In operation, a sample is providedto the input region 120, which, under operation of the electric fieldscreated via the first electrode 110, second electrode 112 and trapelectrode 122 cause the electrophoretic movement of the chargedmacromolecules. Optionally, an additional electrode may be locatedwithin the solution phase region 114, 115.

FIG. 6 shows a perspective view of a first electrode 130, a secondelectrode 132 and a control electrode 134. Generally, the firstelectrode 130 may be identified as the cathode and the second electrode132 the anode, though those terms may be interchanged depending on thepolarity of the connections. The control electrode 134 may be spacedequidistant between the first electrode 130 and second electrode 132,though optionally it may be placed closer to the electrodes 130, 132,most preferably to the cathode 130. Variation of the potential appliedto the control electrode 134 may be used to modulate the flow of chargedmacromolecules within the region between the first electrode 130 andsecond electrode 132. In one mode of operation, the sample is placedbetween the first electrode 130, the cathode, and the control electrode134. The net flow of negatively charged materials is from the cathode tothe anode (second electrode 132). If the control electrode 134 is madeneutral, or even slightly negative, negatively charged materials, suchas DNA, would flow in a direction from the cathode to the anode. Once adesired fraction of the DNA passes through or by the control electrode134, the control electrode 134 may be made more negative, thereby aidingthe motion of the DNA towards the second electrode 132 and repellingundesired material which remains between the control electrode 134 andthe first electrode 130 (cathode).

FIG. 7 shows a perspective view of electrodes advantageously used in abuncher structure. A first electrode 140 and a second electrode 142 arearranged as cathode and anode, respectively (though the terminology maybe reversed). A first control electrode 144 and a second controlelectrode 146 are disposed between the cathode and anode. Bunching ofcharged macromolecules between the first control electrode 144 and thesecond control electrode 146 may be achieved by applying a potential tothe first control electrode 144 so as to accelerate the speed of transitof charged materials relatively closer to the first control electrode144 than to the second control electrode 146. The second controlelectrode 146 is biased so as to retard the speed of charged materialswhich are relatively closer to the second control electrode 146 than tothe first control electrode 144. Since the rate of diffusion of chargedmacromolecules in a solution phase environment is significant comparedto the transit time through the chamber (e.g., the region definedbetween the first electrode 140 and second electrode 142), the bunchingprocess serves to localize the desired charged materials within asmaller chamber, counteracting the effects of diffusion. In one mode ofoperation, once the desired amount of DNA passes the first controlelectrode 144, that control electrode may be placed at a negativepotential which serves to further cause the negatively charged materialstowards the second electrode 142 (anode). While FIG. 7 shows a4-electrode arrangement, a buncher may be formed from the structure ofFIG. 6, by operation of the electrodes in a manner described above.

Broadly stated, this aspect of the invention involves a method forselective isolation of desired charged biological materials fromundesired charged biological materials in a electrophoretic systemhaving a solution phase region, the method including at least the stepof applying a repulsive potential to a first electrode so as toaccelerate motion of the desired charged materials which are relativelycloser to the first electrode than to the second electrode, and applyinga repulsive potential to a second electrode so as to decelerate motionof the desired charged materials which are relatively closer to thesecond electrode than to the first electrode. Such operation results ina spatial distribution of the desired charged materials between thefirst and second electrodes is reduced.

FIGS. 8 and 9 show two implementations in plan view of an integratedsample preparation system of this invention. For convenience, commonlyidentified structures in FIGS. 8 and 9 will be labeled with the samereference numerals. FIG. 8 shows a system in which the tap 166 isdisposed downstream from a protein trap region 162. FIG. 9 shows asystem in which the protein trap chamber 162 is downstream of the tap166.

A purification chamber 150 has disposed at the end thereof a firstelectrode 152 and a second electrode 154. A sample addition port 156 maycomprise an input region of the purification chamber 150. Preferably,the sample addition port 156 comprises a liquid interconnect or coverseal (e.g., luer lock, face seal, slide seal). Optionally, the sampleaddition port 156 includes a filter, such as a 0.2 micron filter. Thefilter optionally serves the function of debris removal and may alsoprovide some shearing of DNA which will reduce the viscosity of the DNA.Optionally, membranes 158 may be utilized at one or both ends of thepurification chamber 150, the principal function of the membranes 158being to isolate the sample material from the electrodes 152, 154.Optionally, a cell lysing device 160 (shown in FIG. 8) is formed in thepurification chamber 150 downstream of the sample addition port 156. Aprotein trap 162 is disposed within the purification chamber 150. In oneembodiment, as shown in FIG. 8, a protein trap 162 is disposed betweenthe sample addition port 156 (and the cell lysing device 160 ifoptionally included) and the tap 166. This option is selectedprincipally if the desired materials for diagnosis have higherelectrophoretic mobility than the protein materials to be trapped. Analternative mode of operation involves the step of causing the proteinsor other undesired materials to have higher degree of mobility than thedesired materials, e.g., DNA. In this mode of operation, a device suchas shown in FIG. 9 may be used and the undesired materials are movedthrough the purification chamber 150 past the tap 166 prior to arrivalat the tap 166 of the desired material, e.g., DNA. Optionally, a proteintrap 162 may be then included between the tap 166 and the secondelectrode 154.

An electrode 164 is preferably disposed within the purification chamber150 at a point adjacent the tap 166 which intersects the purificationchamber 150. FIG. 9 shows a "C-shaped" electrode 165 generally disposedadjacent to and symmetrical with respect to the tap 166. When the DNAband is passing through the chamber defined by the "C" and the bias ofthe C-electrode is then changed to negative (-), the C-shaped electrodeserves to concentrate the DNA or other charged materials containedwithin the space defined by the "C" and to provide a focusing of thecharged materials within that region, while further repelling undesiredmolecules outside the C-region. Additionally, when a positive (+)potential is switched to a electrode located in line with the openportion of the "C", the entire band of DNA with the "C" is focused andpropelled in the new direction toward the positive electrode location.The C-shaped electrode may be considered as various subparts, which maybe formed as a continuous C-shaped structure or as discreet components.First and second electrode portions 165a are disposed generallyperpendicular to the line connecting the first electrode 152 and thesecond electrode 154. These perpendicular electrodes 165a generallyserve to provide a bunching function. The side electrode portion 165bgenerally provides a sideways or transverse force causing the chargedmaterials contained within the C-shaped region towards the tap 166.

The tap 166 comprises a channel or chamber leading from the purificationchamber 150 to the denaturation chamber 168. Optionally, thedenaturation may be performed by heating, such as through a resistiveheater 170, or by other modes or methods known to those skilled in theart, including, but not limited to: other forms of energy inputsufficient to break the DNA strands or other chemical methods known inthe art. In the preferred embodiment, the width of the tap 166 isgreater than 100 microns, and most preferably on the order of 1 mm.Generally, it is desired to have a medium to low surface-to-volumeratio, the preferred embodiment reducing the amount of surface area fornon-specific binding of sample or other materials to the walls of thedevice. The tap 166 leads to the complexity reduction chamber 172. Oneexample of a complexity reduction chamber is described below inconnection with FIGS. 10-12. Optionally, a valve 174 is disposed betweenthe complexity reduction chamber 172 and the diagnostic assay 176. Inthe preferred embodiment, the diagnostic assay 176 comprises an activeprogrammable matrix electronic device of the type described in thevarious application identified in the related application informationsection, above. Optionally, a disposal path 178 is connected to a wastechamber.

FIGS. 10, 11 and 12 show one embodiment of a complexity reductiondevice. The device 180 comprises a printed circuit board 182 and achamber 200 mounted thereon. The printed circuit board 182 preferablyincludes an edge connector 184 to permit the interfacing of thecomplexity reduction system 180 to control electronics. The edgeconnector 184 includes a plurality of conductive fingers 186 whichcontact corresponding conductive portions in a mating edge connector(not shown). The printed circuit board 182 in conventional manner mayinclude a substrate 188. The printed circuit board 182 includesconductors 190 which are patterned into conductive strips and disposedon the substrate 188. Via holes 192 are optionally formed in the printedcircuit board 182, and preferably, the conduction portions 194 extendinto the via holes 192. A conductive gel, such as a polymer gel, mostpreferably agarose, acrylamide or other conductive polymer, is placedwithin the via holes 192. Optionally, these materials may be cured insitu, the curing optionally enhanced or promoted by application of apotential to the conductor 194. In the preferred embodiment, a chamber200 is attached to the printed circuit board 182. A seal 198 serves toform a hermetic seal between the chamber 200 and the substrate 188.Within the chamber 200, a sample volume 201 is formed to contain asample for complexity reduction. Optionally, an input port and an outputport may be included within the chamber 200 to provide access to thechamber volume 201. Alternatively, the sample may be supplied into thesample volume 201 through an opening at the surface. Within the chamber200, one or more probe areas 202 form the upper portion of the via holes192. The gel 196 preferably fills this space and terminates at thebottom of the sample volume 201. Optionally, disposal or dump areas 204may be included within the chamber 200. Preferably, an index detent 206is provided within the substrate 188. A matching key 208 is preferablyformed on the underside of the chamber 200 to aid in indexing of thechamber 200 relative to the printed circuit board 182. As shown in FIG.12, the chamber 200 may then be matingly engaged with the printedcircuit board 182, with the keys 208 joining with the index detents 206.In operation, the polymer gel 196 includes capture probes. These captureprobes then interact with the target materials in the sample andhybridize thereto. Conductive polymer may be used to fill the vias ofthe complexity reduction device in order to provide a matrix for DNAprobe attachment, and DNA target hybridization and separation. Thepolymer can be mixed with protein bound DNA capture probe prior tofiling the vias of the device in order to introduce polymerfunctionality or covalently bound capture probe may be pre-mixed withthe conductive polymer. Alternatively, the probe may beelectrophoretically transported into the polymer and linked by enzymaticor covalent means in order to provide additional means for attachment tothe polymeric support.

In operation, the target DNA may be placed directly in the sample wellof the complexity reduction device or fluidically or electrophoreticallyintroduced into the sample chamber. The sample can be introduced in oneof several different buffers, including 50 mM sodium borate pH 8, or0.5×TBE. These buffers provide for free field electrophoretic transportof DNA at relatively low ionic strengths. Test results are shown in FIG.17. Also, enhanced hybridization is shown for 0.5×TBE and histidine inFIG. 16. During transport, the electrodes are biased with a positivecurrent and the target DNA is transported electrophoretically into thepolymer filled vias of the device allowing the complementary target DNAto hybridize to the specific capture probe. Next, during the electronicwash procedure, the DNA which is not a specific match to the capture DNAis removed from the vias using a mild negative current. A fluidic washremoves any irrelevant DNA from the sample well and fresh buffer is thenintroduced. The hybridized target DNA then may be dehybridizedelectrophoretically using a strong negative current.

In order to maximize DNA purification, electrophoretic transport,hybridization, electronic wash and dehybridization can be performedusing a variety of electronic settings. For transport and accumulation,the settings include a positive DC current of between 5 to 2,500 μA/mm²per polymer filled via for 10 s to 180 s (preferably: 200 to 500 μA/mm²for 15 to 60 s), a pulsed current of between 5 to 2,500 μA/mm² at a 25to a 75% duty cycle for 15 to 180 s (preferably: 200 to 1,000 μA/mm²,50% duty, 15 Hz for 20 to 180 s), and a reverse linear stair starting atbetween 100 to 500 μA/mm² and ending at 0 to 150 μA/mm² in 15 to 90 s(preferably: starting at 250 μA/mm² and ending at 25 μA/mm² in 90 s). Anelectronic wash is conducted with a negative DC bias between 200 to 300μ/A/mm² or a pulsed current of between 200 and 500 μA/mm² for 15 to 180s. Dehybridization is performed at a negative DC current of 400 to 750μA/mm² for 60 to 420 s.

For purposes of fluorometric detection, target DNA can be labeled with afluorophore, "reverse dot blot" hybridization, FIGS. 16 and 19. Inaddition, the target DNA can be detected after hybridization to thecapture probe by the introduction of a fluorophore labeled strand of DNAcomplementary to a nonhybridized chamber of the target DNA "sandwich"hybridization. Alternatively, the fluorophore label can be incorporatedinto the target DNA which has been amplified by PCR.

FIG. 13 shows a perspective, cutaway view of a vertically disposed DNAor nucleic acid purification device of this invention. An upperreservoir 210 communicates with a lower reservoir 212 via tube 214permitting fluid communication from the upper reservoir 210 to the lowerreservoir 212. The reservoirs 210, 212 are adapted to receive a buffersolution 216. Optionally, the upper reservoir 210 and lower reservoir212 may be formed so as to permit formation of a closed system, such asby causing the bottom 218 of the upper reservoir 210 to sealinglycontact the top 220 of the lower reservoir 212. Alternatively, thesystem may be an open system. The upper reservoir 210 contains a firstelectrode 222 and the lower reservoir contains a second electrode 224.The first electrode 222 and the second electrode 224 may be referred toas the cathode and anode, respectively, though those terms may beinterchanged given the polarity of operation. The tube 214 preferablyhas an inner diameter which is smaller than the inner diameter of thereservoirs 210, 212. The tube 214 contains a polymer 226. The polymer226 is a molecular sieve which comprises a differential mobilitychamber. Materials which provide differential mobility for chargedbiological macromolecules include materials such as agarose andpolyacrylamide.

In the preferred embodiment, the differential mobility material is acast 1.5% agarose gel in 50 mM histidine, formed on a supportingmembrane 228 in tube 214. Optionally, the polymer region 226 is disposedadjacent a membrane 228. Preferably, the membrane 228 is porous, e.g., 5micron pore size, and serves in part to provide a support for theformation of the polymer 226. A chamber 230 is disposed adjacent thepolymer 226. Preferably, the chamber 230 has a volume which is less thanthat of the conductive polymer 226, e.g., preferably being approximately50% or less in volume, and most particularly approximately 1/3 or lessin volume than the polymer 226. If the chamber 230 has a reduced volumerelative to the polymer 226, a reduced inner diameter chamber 232 may beformed in the tube 214. This reduced inner diameter region 232advantageously forms a ledge 234 providing an annular region on whichthe membrane 228 may disposed. Optionally, a second membrane 236 maydefine a portion of the boundary of chamber 230. The second membrane 236preferably constitutes a molecular weight cut-off membrane, such as anultrafiltration membrane, which serves to retain the DNA within thechamber 230, but passes smaller materials, such as proteins subjected toproteinase K. Such ultrafiltration membranes include those formed fromcellulose acetate or cellulose. In the preferred embodiment, theelectrodes are disposed generally parallel to the membrane (236), and ata distance from the membrane that is equal to, or greater than, theheight of the collection chamber (230).

In operation, a sample is previously lysed such as by motion of glassbeads acting on cells of the sample. Additionally, the sample ispreferably subject to shearing, such as by movement through a relativelynarrow aperture, such as an aperture of diameter of 250 microns. Thesample is preferably subject to a treatment step which reduces the sizeof the proteins or other undesired materials so as to increase theirdifferential mobility relative to the desired material, e.g., DNA. Forexample, addition of proteinase K may reduce the size of proteins, suchas to 20,000 daltons or less. This application of proteinase K may bedone at room temperature, though performing it at an elevatedtemperature, e.g., 37° C. to 50° C., increases the rate of reaction. Inthe preferred embodiment, the lysate cells are digested with 250 μg/mlproteinase K, 0.5×TBE 50 mM EDTA buffer (37-50° C.) with a total volumeof 50 μl. Next, a densification agent, such as sucrose, e.g., preferably5%-20%, and most preferably substantially 8-10% serves to densify thesample. Optionally, the densified material may be combined with a dye,such as bromphenol blue. The densified, pre-prepared sample is theninjected or placed above the polymer 226, such as by use of a syringe.Use of the densified sample serves to locate and concentrate the sampleimmediately above the polymer 226. The system is then operated to causethe electrophoretic motion of charged materials. The system is activatedfor a time to permit the DNA to migrate into the polymer 226, and topermit the reduced size proteins to substantially traverse the polymer226 into the chamber 230 and lower reservoir 212. In the preferredembodiment, the sample is run into the gel for six to eight minutes, ata current of 2.25 mA, with a 1,000 V limit. The cathode additionallyserves to permit attraction and destruction of the proteinase K andother positively charged materials. Optionally, fresh buffer 216 isprovided in the lower reservoir 212, and the second membrane 236 may beadded at this time (though it may be initially included within thedevice). In the preferred embodiment, the membrane 236 is a 25 kDmolecular weight cutoff membrane. Next, the DNA is eluted from thepolymer 226 into the chamber 230. In the preferred embodiment, thesample is eluted out of the polymer into the elution chamber 230 forapproximately two minutes, with the 1,000 V limit. The DNA is thenextracted from the chamber 230, such as by piercing or providing a portand valve arrangement.

Though the system of FIG. 13 is shown in a vertical arrangement, it maybe performed in a horizontal arrangement. The vertical arrangementpermits a constant contact area between the sample solution 236 and thepolymer 226. Further, the use of the densification agent permits thelocalization of the sample solution 236 immediately adjacent the polymer226, reducing the time necessary for the sample solution 236 to reachthe polymer 226. In a horizontal arrangement, as shown in FIG. 14, apolymer (or gel or membrane) dam can be used to maintain the separationof the sample from the electrode buffer to prevent mixing.

FIG. 14 shows a plan view of an integrated device including a samplepreparation, complexity reduction, diagnostic chamber and disposalchamber. FIG. 14A shows a cross-sectional view of FIG. 14 along the lineA-A'. A support member 240, such as a printed circuit board, preferablyserves to support the various components described below. Optionally, anedge connector 242 may provide electrical connection to controlelectronics, as discussed previously in connection in FIGS. 10-12. Asample preparation chamber 244 preferably includes a first buffercontaining region 246 which also is in electrical communication with anelectrode. A material 248 such as a gel or other conductive material isdisposed between the buffer region 246 and the input port 250. In thepreferred embodiment, the input port 250 includes a cover, which may beoptionally removed for sample input or which can be sealed and piercedonce the sample is placed within the sample port 250. A DNA trap 252 isdisposed between the input port 250 and the protein trap 260.Preferably, the DNA trap 252 narrows or constricts as materials areelectrophoresed through the DNA trap 252. In one embodiment, a slopedupper portion 254 and inwardly sloped side walls 256 serve to form aconstriction at the gel boundary 258. Such a structure provides aconcentrating effect. Preferably, the protein trap 260 and the bufferspace 264 are formed in a y-shaped manner. The protein trap 260 thencontacts the buffer space 262 which includes an electrode.

The sample preparation structure 244 operates generally as follows.First, a sample is placed in the input port 250. Electrophoretic actionof the electrodes causes conduction of the charged macromolecules from adirection connecting the buffer region 246 towards the buffer region262. In the preferred embodiment, the sample is subject to aprepreparation step which lyses the cells and digests the protein,resulting in proteinaceous material which has a relatively highermobility than the DNA through the DNA trap 252. Thus, as electrophoresiscontinues, the protein materials arrive at the protein trap 260 prior tothe arrival of the DNA. After the proteins arrive at the protein trap260, the electrode and buffer region 262 is turned neutral and theelectrode and buffer region 264 is biased from neutral to positive. TheDNA moving through the DNA trap 250 are then attracted by a positivepotential applied to the buffer space 264. In this manner, the proteinsare shunted to the protein trap 260 whereas the DNA, most likely at alater time, are shunted to the chamber port 266. The DNA concentrationvolume is shown in FIG. 14A as that portion beyond the gel boundary 258within the buffer space 264.

In one aspect of the invention, the buffer space 264 includes as aninput a chamber port 266 coupled to one end of the buffer space 264 anda second port or inlet port 268 which is fluidically coupled to theopposite end of the buffer space 264, as shown in FIGS. 14 and 14A beingconnected by a bound tube 270 to the buffer space 264. In operation, theinlet port 268 and chamber port 266 may receive the same or differentliquid or gas, such as a buffer, a reagent or an air slug. By operationof the materials provided to the inlet port 268 and chamber port 266, ahydraulic or pneumatic ram may result. For example, if the buffer space264 contains DNA which has been eluted from the DNA trap 252 into thebuffer space 264, that material may be forced into the connector 272 byforcing fluid, e.g., buffer, into the inlet port 268 causing the DNA tomove into the connector 272. Advantageously, air slugs may be used toseparate various fluidic materials. Additionally, fluid may be withdrawnfrom the inlet port 266, 268 to cause the movement of other materials ina direction generally opposite to the normal processing flow direction.

The structure shown in FIGS. 14 and 14A additionally includes theconnector 272 being in communication with the complexity reductionregion 274. An exterior containment vessel 276 defines the outer edge ofthe complexity reduction region 274. One or more probe areas 278, havingthe structure and function described previously with respect to FIGS.10-12, may be utilized. Similarly, one or more dump areas 280 may bedisposed within the complexity reduction region 274 as described inconnection with FIGS. 10-12. Preferably, a volume reduction region 282connects the complexity reduction region 274 to the diagnostic chamber284. The reduced volume region 282 serves a concentrating function. Thestructure and function of the diagnostic portion 284 may include anyknown diagnostic, but preferably includes an electronically enhancedhybridization/dehybridization device such as described in "ActiveProgrammable Electronic Devices for Molecular Biological Analysis andDiagnostics", U.S. Ser. No. 08/146,504, filed Nov. 1, 1993, incorporatedherein by reference. Preferably, a connector 288 provides fluidcommunication to a waste chamber 286. The waste region 286 preferably isa fully contained volume so as to avoid biological contamination.

While the methods and devices herein have generally been described as aserial system, e.g., a sample preparation section, a complexityreduction section and an assay section, some or all of the stages may beperformed in a parallel or multiplexed format. In one embodiment, two ormore parallel sets, each comprising a sample preparation region, acomplexity reduction chamber and an assay may be used. As an alternativeembodiment, two or more sets of sample preparation sections may provideoutput to a smaller number, e.g., one, complexity reduction region,preferably followed by an assay. Alternatively, two or more sets of asample preparation section and a complexity reduction chamber mayprovide their outputs to a smaller number, e.g., one, assay. Othervariations and combinations consistent with the invention will beapparent to those skilled in the art.

Further, while the description in the patent refers often to DNA, itwill be understood that the techniques may be applied to RNA or othercharged macromolecules, when consistent with the goals and functions ofthis invention. When the inventive methods and apparatus are used forRNA at the time of lysis, the user would preferably add RNAse inhibitorsand RNAse free DNAse to remove DNA. The remainder of the purificationprocess would follow as before. The isolation of poly A RNA, whichincludes most of the mRNA, and removal of ribosomal RNA is preferablyperformed in the complexity reduction chamber. Oligo dT probes couldoptionally be used to bind the polyA RNA during electronic hybridizationand unhybridized RNA, mostly ribosomal RNA, would be removed. To releasethe poly A RNA, electronic dehybridization could preferably be employed.Similarly, a specific sequence of mRNA could be isolated by using probescomplementary to that sequence in the complexity reduction device.Electronic hybridization would be performed as for DNA targets and theunhybridized, irrelevant RNA would be removed. The desired mRNA specieswould be eluted from the probes by electronic dehybridization.

The sources and control systems for supply of the potential, currentand/or power to any of the electrodes of these inventions may beselected among those known to persons skilled in the art. The voltageand/or current may be supplied with either fixed current or fixedvoltage, with the other variable permitted to float, optionally subjectto limits or maximum values. The control system may be analog ordigital, and may be formed of discrete or integrated components,optionally including microprocessor control, or a combination of any ofthem. Software control of the systems is advantageously utilized.

EXPERIMENTAL DATA Example 1 Comparison of FIG. 13 Device to Qiagen

The relative performance of a device as shown in FIG. 13 was compared tothe prior art Qiagen system. For the comparison, the preparation andoperation of the FIG. 13 device was as follows.

First, 50 mL of a stationary phase suspension culture of Staphylococusaureus cells was pelleted and resuspended in 1000 μl of 0.5×TBE, 50 mMEDTA, and lysed by vortexing in the presence of glass beads. Then, RNAand protein were fragmented by digesting with 50 μg/ml RNAse and 250μg/mL proteinase K for 40 minutes at 50° C. Next, the sample density wasincreased by the addition of 5 μl of 40% sucrose to 20 μl sample, whichis roughly equivalent to 10⁹ cells, to achieve a final concentration of8%. Prior to loading the sample, the device was filled with 50 mMhistidine and then, 25 μl of sample was then loaded in the samplesolution zone 236. In other experiments, volumes as high as 100 μl havebeen used with similar results. Next, the electrodes were connected to apower supply and current was sourced at 2.5 mA. In other experiments,currents as low as 1 mA have been used with similar results except thattransport was slowed. After 3 minutes, the power was turned off, thebarrel was removed, and a cellulose acetate membrane with a 25 kDmolecular weight cutoff was inserted as second membrane 236. Thesolution was also removed and fresh 50 mM histidine was added to thedevice. After reassembly, the power was turned on and the sample waseluted into chamber 230 formed by the cellulose acetate membrane 228which supports the gel and the 25 kD cutoff cellulose acetate membrane236. To remove the eluted sample, the power is turned off, the barrelwas removed, a pipette/syringe is used to puncture the 25 kD cutoffmembrane and the sample solution was withdrawn. The total time for allof the electrophoretic steps was less than 5 minutes. The sample wasanalyzed by agarose gel electrophoresis and spectrophotometry. Gelelectrophoresis showed that the DNA was approximately 20 kbp in lengthand that the yield was approximately 20%.

For comparison, a crude sample prepared by the same method of lysis anddigestion was processed in the Qiagen device. The Qiagen device wasoperated in accordance with its instructions. A comparison to resultsobtained with the Qiagen device is as follows.

Optical density readings were performed and the ratio of the opticaldensity at 260 nanometers to 280 nanometers was determined. The deviceof FIG. 13 provided a ratio of from 1.6 to 1.8 on various operations, incontrast, the Qiagen device providing a result of less than 1.3 with thesame material (the Qiagen literature does state that a ratio of 1.7 to1.9 can be obtained). Thus, in the actual testing on this sample, thedevice of FIG. 13 provided purified DNA. Secondly, the transport of theprepared sample on a microelectronic chip constructed as disclosed in"Active Programmable Electronic Devices for Molecular BiologicalAnalysis and Diagnostics", Ser. No. 08/146,504, filed Nov. 1, 1993, wasbetter than the transport on the same device of the Qiagen preparedmaterial. It is believed that the Qiagen material has a relatively highresidual salt content, and that therefore the transport on the chip waspoorer than in the case of transport of material prepared by the methodand structure of FIG. 13. FIG. 15 shows test results for transport ofsamples prepared by the device of this invention as compared to sampleprepared by the Qiagen device. Third, sample preparation wassignificantly faster with the FIG. 13 device, approximately 5 minutes(6.5 minutes in one test) in comparison to over two hours for the Qiagendevice. Finally, the yields for both methods were similar, approximately20%.

Example 2 Performance of FIG. 1 Device

A device was prepared with the same structural components as FIG. 1,though its actual shape was as disclosed here. The purification devicewas constructed from polymethacrylate (PMA) generally as shown inFIG. 1. To allow the insertion of different materials 22, 16 and 24, thedevice is assembled from separate sections of PMA whose ends meet at thelines indicated by the spacer region compartment 16. The sections areheld together by screws which run longitudinally. Electrodes made of Ptwires were attached to electrodes 26, 28. The wires protruded intoelectrode chambers 18, 20. Electrode chambers 18, 20 were each filledwith 300 μl TAE, 250 mM HEPES. An Elutrap ultrafiltration celluloseacetate membrane from Schleicher and Schuell was inserted between PMAsections at position membrane 24 and a cellulose acetate membrane with a25 kD cutoff from Sialomed was inserted at membrane position 22 toseparate the electrode chambers 18, 20 from the sample chambers 12, 14.The sample chambers 12, 14 were filled with 75 μl each of TAE, 250 mMHEPES. Although different membranes have been inserted at spacercompartment 16 to achieve separation of DNA from protein, in thisexperiment, spacer compartment 16 contained an Immobilon P membrane(PVDF with 0.45 μm pores) from Millipore. The membrane was wetted withmethanol and soaked in 1×TAE, 250 mM, 0.1% Triton x 100 prior to loadinginto spacer compartment 16. A crude sample, a 162 μl of a mixture of aprotein, 48.5 μg of Bodipy Fluorescein labeled BSA, and 0.79 μg of aBodipy Texas Red labeled 19 mer in 1×TAE, 250 mM HEPES was loaded intochamber 12. After the addition of the sample, first electrode 26 wasbiased negative and second electrode 28 was biased positive for 2.75minutes at 10 mA. As a result, the sample was electrophoreticallytransported to spacer compartment 16. The labeled DNA passed through themembrane and was collected on the other side of the membrane in rightcentral chamber 14. The amounts of DNA and BSA were determined byfluorimetry. The results show that the yield of DNA was 40% an that 78%of the BSA was removed.

Example 3 Performance of Complexity Reduction Device

In Group A Streptococcal (GAS) experiments, FIGS. 18 and 19, the polymeris pre-mixed with a 20 μM concentration of a 25 mer streptavidin-biotinbound capture probe. In GAS experiments, FIGS. 18 and 19, 40 μL aliquotsof target DNA are placed into the sample well in 50 mM histidine.Electrophoretic transport was conducted using a linear stair starting at250 μA/mm² and ending at 25 μA/mm² an the electronic wash was conductedusing a 250 μA/mm² pulse for 45 s and 120 s, respectively.Dehybridization, FIG. 18, was conducted at 400 μA/mm² for 150 s in0.5×TBE. Results from FIG. 18 show a purification ratio of approximately33-fold (100:3) and a recovery of 82% of the pure hybridized target.FIG. 19 shows purification of a specific target in increasing ratios ofirrelevant DNA with a purification ratio of 3.4 fold in the presence ofa 200,000 fold mass excess of irrelevant material.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

We claim:
 1. An apparatus for active biological sample preparation adapted to separate desired materials from undesired materials comprising:a first sample chamber, adapted to receive a buffer solution, a second sample chamber, adapted to receive a buffer solution, a spacer compartment disposed between the first sample chamber and the second sample chamber, the spacer compartment providing a trap having a differential effect on the desired materials versus undesired materials, a first electrode adapted for electrical contact with the buffer solution when located within the first sample chamber, and a second electrode adapted for electrical contact with the buffer solution when in the second sample chamber, characterized by having a protective layer between the first sample chamber and the first electrode, thereby forming a first electrode chamber within the first sample chamber, wherein the protective layer is a membrane.
 2. The apparatus for active biological sample preparation of claim 1 wherein the trap is hydrophobic.
 3. The apparatus of claim 1 for active biological sample preparation wherein the trap is a protein trap.
 4. The apparatus of claim 3 wherein the protein trap is polyvinylidene diflouride (PVDF).
 5. The apparatus of claim 3 wherein the protein trap is nitrocellulose.
 6. The apparatus of claim 1 for active biological sample preparation wherein the trap is a DNA trap.
 7. The apparatus of claim 1 further comprising a third electrode disposed proximal to the trap.
 8. The apparatus of claim 7 for active biological sample preparation wherein the third electrode and trap comprise a metal-coated filter material.
 9. The apparatus of claim 1 further comprising a third electrode formed integral with the trap.
 10. The apparatus of claim 1 for active biological sample preparation further comprising a control system operatively coupled to the electrodes.
 11. The apparatus of claim 1 for active biological sample preparation wherein the spacer compartment has a length, a width, and a depth defining a volume.
 12. The apparatus of claim 11 for active biological sample preparation wherein the volume of the spacer compartment is less than 50% of a volume of the first sample chamber.
 13. The apparatus of claim 12 for active biological sample preparation wherein the volume of the spacer compartment is 30% or less of the volume of the first sample chamber.
 14. The apparatus of claim 13 for active biological sample preparation wherein the volume of the spacer compartment is 25% or less of the volume of the first sample chamber.
 15. The apparatus of claim 1 for active biological sample preparation wherein the length of the sample chamber is 20% or less of the distance between the first electrode and the second electrode.
 16. The apparatus of claim 15 for active biological sample preparation wherein the length of the sample chamber is 10% or less of the distance between the first electrode and the second electrode.
 17. The apparatus of claim 16 for active biological sample preparation wherein the length of the sample chamber is 5% or less of the distance between the first electrode and the second electrode.
 18. The apparatus of claim 1 for active biological sample preparation wherein the length of the sample chamber is 5 nm or less.
 19. The apparatus of claim 18 for active biological sample preparation wherein the length of the sample chamber is 4 mm or less.
 20. The apparatus of claim 19 for active biological sample preparation wherein the length of the sample chamber is from approximately 1 to approximately 2 mm.
 21. The apparatus of claim 1 for active biological sample preparation wherein the membrane is an ultrafiltration membrane.
 22. The apparatus of claim 1 for active biological sample preparation wherein the membrane is a cellulose acetate membrane.
 23. The apparatus of claim 1 wherein the first electrode chamber has a volume equal to at least 10 times a volume of at least one of the first or second sample chambers.
 24. The apparatus of claim 1 for active biological sample preparation wherein the first sample chamber is disposed vertically above the spacer compartment.
 25. A method for active biological sample preparation of a sample comprising a collection of materials including desired materials and undesired materials having differential charge-to-mass ratios, whereinthe desired material comprises specific cells and the undesired material comprises a remainder of the sample, the separation being achieved in an apparatus as defined in claim 1 comprising the steps of:providing the sample to the first sample chamber of the apparatus, electrophoresing the sample within the apparatus to affect net differential migration between the desired material and the undesired material, whereby the desired material is located within the trap and the undesired material is in the second chamber, removing the desired material from the, apparatus, whereby relatively purified desired materials are prepared.
 26. The method of claim 25 wherein the desired material is eluted from the trap.
 27. A method for arrive biological sample preparation of a sample comprising a collection of materials including desired materials and undesired materials having differential charge-to-mass ratios whereinthe desired material comprises at least one of DNA and RNA and the undesired material comprises protein, the separation being achieved in an apparatus as defined in claim 1 comprising the steps of:providing the sample to the first sample chamber of the apparatus, electrophoresing the sample within the apparatus to affect net differential migration between the desired material and the undesired material, whereby undesired material is located within the trap and the desired material is in the second chamber, removing the desired material from the apparatus, whereby relatively purified desired materials are prepared.
 28. The apparatus for biological sample preparation of claim 1 wherein the trap is a RNA trap.
 29. The apparatus for biological sample preparation of claim 1 wherein the trap is a dipstick.
 30. A method for active biological same preparation of a sample comprising a collection of materials including desired materials and undesired materials having differential charge-to-mass ratios, the separation being achieved in an apparatus as defined in any of claims 1-19 and 21-24 comprising the steps of:providing the sample materials to the first sample chamber of the apparatus, electrophoresing the sample within the apparatus to affect net differential migration between the desired material and the undesired material whereby one of the desired or undesired materials is located within the trap and the other material is in the second sample chamber, removing the desired material from the apparatus, whereby relatively purified desired materials are prepared.
 31. The method of claim 3 wherein after at least a portion of the desired material has contacted the trap, a potential is applied to the undesired materials in at least one of the first or second sample chambers so as to repel them from the trap.
 32. The method of claim 30 for active biological sample preparation wherein the sample is first electrophoretically transported through the first sample chamber prior to contacting the trap.
 33. The method of claim 32 for active biological sample preparation wherein the desired material is located within the trap.
 34. The method of claim 33 for active biological sample preparation comprising the additional step of removing the desired material and trap from the apparatus.
 35. The method of claim 33 for active biological sample preparation comprising the additional step of removing the desired material from the trap.
 36. The method of claim 35 for active biological sample preparation comprising the additional step of removing the desired material from the trap to one of the first or second sample chambers.
 37. The method of claim 36 for active biological sample preparation wherein the desired material is removed from the trap to the first sample chamber.
 38. The method of claim 36 for active biological sample preparation wherein the desired material is removed from the trap to the second sample chamber.
 39. The method of claim 32 for active biological sample preparation wherein the undesired material is located within the trap.
 40. The method of claim 39 for active biological sample preparation comprising the additional step of removing the desired material from the system by contact with solution in one of the first or second sample chambers.
 41. The method of claim 39 for active biological sample preparation wherein the desired materials are electrophoresed through the trap prior to the undesired materials being trapped in the trap.
 42. The method of claim 30 for active biological sample preparation wherein the sample is initially placed in the first sample chamber immediately adjacent the trap.
 43. The method of claim 42 for active biological sample preparation wherein the desired material is retained in the trap and the undesired material is electrophoresed through the trap into the second sample chamber.
 44. The method of claim 43 for active biological sample preparation wherein the desired material is eluted from the trap after the undesired materials are electrophoresed through the trap.
 45. The method of claim 44 for active biological sample preparation wherein the desired materials are eluted into the second sample chamber.
 46. The method of claim 30 for active biological sample preparation wherein the sample is subject to a proteinase step prior to providing the sample materials to the first sample chamber of the device.
 47. The method of claim 30 for active biological sample preparation wherein the sample is subject to a densification step prior to electrophoretic transport of charged materials.
 48. The method of claim 47 for active biological sample preparation which further comprises the step of subjecting the sample to a proteinase prior to subjecting the sample to the densification step.
 49. The method of claim 30 for active biological sample preparation wherein the sample is subject to a proteinase prior to electrophoretic transport of charged materials.
 50. A device for purification of DNA from a sample including proteinaceous material having a charge-to-mass ratio which is greater than that of the DNA, the device comprising:an upper reservoir adapted for receipt of a buffer solution, and for receipt of a sample solution containing DNA and proteinaceous material, a collection chamber, disposed downstream of the upper reservoir, adapted for receipt of a buffer solution and for receipt of the DNA, characterized by having a membrane defining at least a portion of the collection chamber, a polymer, disposed upstream and adjacent to the collection chamber, wherein the polymer is in a fixed position relative to the collection chamber, electrodes adapted for electrical contact with the buffer solution to provide electrophoretic movement of the DNA and proteinaceous material through the polymer and collection chamber, wherein the electrodes are disposed in a direction which is parallel to the membrane, and the distance between the electrodes and the membrane is equal to or greater than the height of the collection chamber.
 51. The device of claim 50 for purification of DNA wherein the membrane is an ultrafiltration membrane.
 52. The device claim 50 for purification of DNA wherein the membrane comprises a molecular weight cut-off membrane.
 53. The device of claim 50 for purification of DNA wherein the molecular weight cut-off membrane retains the DNA within the collection chamber.
 54. The device of claim 50 for purification of DNA wherein the collection chamber has a volume which is smaller than a volume of the polymer.
 55. The device of claim 54 wherein the collection chamber volume is less than or equal to half the volume of the polymer.
 56. The device of claim 55 for purification of DNA wherein the volume of the collection chamber is equal to or less than 1/3 the volume of the polymer.
 57. The device of claim 50 for purification of DNA further comprising a membrane between the polymer and the collection chamber.
 58. The device of claim 57 for purification of DNA wherein the membrane has a pore size which permits transport of both the DNA and proteinaceous materials through the membrane.
 59. The device of claim 50 for purification of DNA further comprising a lower reservoir adapted to receive a buffer solution and disposed beneath the collection chamber.
 60. The device of claim 50 wherein the polymer is a molecular sieve.
 61. The device of claim 60 wherein the molecular sieve is acrylamide.
 62. The device of claim 50 further comprising a buffer.
 63. The device of claim 62 wherein the buffer is histidine.
 64. The device of claim 50 wherein the polymer has a thickness in the direction of migration of substantially 10 to 20 mm.
 65. A method for purification of DNA from a sample mixture including DNA and other charged materials, including certain materials having a higher charge-to-mass ratio than the DNA, in a device as defined in any of claims 50 to 64, the method comprising the steps of:placing the sample mixture in the upstream buffer chamber above the polymer, activating the electrodes so as to electrophorese the sample mixture into the polymer for a time sufficient to cause the undesired material to substantially move through the polymer, and eluting the DNA into the collection chamber.
 66. The method of claim 65 for purification of DNA from a sample mixture which further comprises the step of densification of the sample mixture prior to electrophoresing the sample mixture.
 67. The method of claim 66 for purification of DNA from a sample mixture wherein the densification step includes the addition of sucrose.
 68. The method of claim 62 for purification of DNA from a sample mixture which comprises the further step of lysing of cells prior to the placement of the sample mixture in the upstream buffer chamber.
 69. The method of claim 68 for purification of DNA from a sample mixture which comprises the further step of shearing the sample prior to placement of the sample mixture in the upstream buffer chamber.
 70. The method of claim 65 for purification of DNA from a sample mixture which comprises the further step of subjecting the sample to a proteinase prior to electrophoresing the sample mixture.
 71. The method of claim 70 for purification of DNA from a sample mixture wherein the sample mixture is subject to an RNAse step prior to said proteinase step.
 72. The method of claim 71 wherein the proteinase is proteinase K.
 73. The method of claim 65 for purification of DNA from a sample mixture which comprises the further step of changing the buffer solution prior to the step of eluting the DNA into the collection chamber.
 74. The method of claim 65 for purification of DNA from a sample mixture which comprises the further step of adding a membrane to the collection chamber prior to eluting the DNA into the collection chamber.
 75. The method of claim 65 for purification of DNA from a sample mixture which comprises the further step of extracting the DNA from the collection chamber.
 76. The method of claim 75 for purification of DNA from a sample mixture wherein the DNA is extracted from the collection chamber by piercing of the collection chamber.
 77. The method of claim 75 for purification of DNA from a sample mixture wherein the DNA is extracted from the collection chamber through a valve. 